Incorporation of (2S,3S) and (2S,3R) β-methyl aspartic acid into RGD-containing peptides

Article abstract of DOI:10.1016/S0968-0896(02)00206-7

We report the synthesis and biological activity of a series of side-chain-constrained RGD peptides containing the (2S,3R) or (2S,3S) β-methyl aspartic acid within the RGD sequence. These compounds have been assayed for binding to the integrin receptors α_IIbβ₃ and α_vβ₃ and the results demonstrate the importance of the side-chain orientation of this particular residue within the RGD sequence. Based on our findings, the (2S,3S) β-methylated analogues of our RGD sequences maintain their binding potency to the integrin receptors while the (2S,3R) β-methylated analogues exhibit a drastically reduced binding affinity. Our studies demonstrate that the three-dimensional orientation of the aspartyl side chain is a very important parameter for integrin binding and that small changes that affect the side-chain orientations give rise to drastic changes in binding affinity. These results provide important information for the design of more potent RGD mimetics. Copyright

Full text of DOI:10.1016/S0968-0896(02)00206-7

Bioorganic & Medicinal Chemistry 10 (2002) 3331–3337
Incorporation of (2S,3S) and (2S,3R) ꢀ-Methyl Aspartic
Acid into RGD-Containing Peptides
Silke Schabbert,a Michael D. Pierschbacher,b Ralph-Heiko Matternb,*
and Murray Goodmana,*
aDepartment of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093-0343, USA
bIntegra LifeSciences Corporation, Corporate Research Center, San Diego, CA 92121, USA
Received 18December 2001; accepted 16 April 2002
Abstract—We report the synthesis and biological activity of a series of side-chain-constrained RGD peptides containing the (2S,3R)
or (2S,3S) b-methyl aspartic acid within the RGD sequence. These compounds have been assayed for binding to the integrin
receptors aIIbb3 and avb3 and the results demonstrate the importance of the side-chain orientation of this particular residue within
the RGD sequence. Based on our findings, the (2S,3S) b-methylated analogues of our RGD sequences maintain their binding
potency to the integrin receptors while the (2S,3R) b-methylated analogues exhibit a drastically reduced binding affinity. Our
studies demonstrate that the three-dimensional orientation of the aspartyl side chain is a very important parameter for integrin
binding and that small changes that affect the side-chain orientations give rise to drastic changes in binding affinity. These results
provide important information for the design of more potent RGD mimetics.
# 2002 Elsevier Science Ltd. All rights reserved.
Introduction
processes such as angiogenesis, and adhesion of osteo-
clasts to the bone matrix. Antagonists to this receptor
could be useful in the treatment of osteoporosis, dia-
betic retinopathy and cancer.9
Integrins are a family of heterodimeric glycoprotein1,2
complexes that regulate cell–cell and cell–matrix inter-
action.3 These transmembrane receptors mediate a
variety of cell adhesion events and signal transduction
processes and are involved in an array of pathological
events such as tumor metastasis, angiogenesis, throm-
bosis and osteoporosis. Integrin receptors consist of an
a and b subunit which are non-covalently linked and
many of these receptors bind their natural ligands
through an RGD sequence.4À6
A number of potent and specific antagonists to aIIbb3
10
and avb3 have been developed and the three-dimen-
sional structure of some of these compounds in solution
have been studied. From these studies the backbone con-
formations of avb3 and aIIbb3 specific ligands have been
postulated.11 As part of our ongoing efforts to optimize
integrin antagonists and develop non-peptide analogues
we have synthesized a series of peptides incorporating
(2S,3S) and (2S,3R) b-methylated aspartic acid residues.
These studies were carried out to constrain the Asp side
chain and gain information on the structural requirements
for binding of this side chain. We have chosen some of
our peptide pharmacophores for these studies. In parti-
cular, we have chosen the avb3 selective pharmacophores,
Arg-Gly-Asp-Asp-Val, Arg-Gly-Asp-Asp-(tBuG) and
Arg-Gly-Asp-Tyr(Me)-Arg as well as the non-selective
but potent Arg-Gly-Asp-Thr-Tic pharmacophores con-
strained by either disulfide bridges or a head to tail
cyclization through a 3-aminomethyl benzoic acid
(Mamb) residue. These pharmacophores and their
activities has been described earlier.10b,12
Among the integrin receptors, two receptors that were
among the first to be discovered have emerged as being
of particular interest7 and have been extensively studied,
the fibrinogen receptor, aIIbb3, and the vitronectin
receptor, avb3. The aIIbb3 is important for platelet
aggregation and antagonists to this receptor have ther-
apeutic potential as antithrombotic agents.8 The vitro-
nectin receptor is involved in a number of biological
*Corresponding authors. Fax:+1-858-534-0202; e-mail: mgoodman@
usdc.edu (M. Goodman); Fax: +1-858-535-8269; e-mail: rmattern@
integra-LS.com (R.-H. Mattern)
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00206-7

3332
S. Schabbert et al. / Bioorg. Med. Chem. 10 (2002) 3331–3337
We now report the syntheses and binding results of
these cyclic RGD peptide analogues containing
enantiomeric pure b-methylated (2S,3S) and (2S,3R)
aspartic acid building blocks.
without alkylation or epimerization of the a-carbon. A
mixture of both b-methylated diastereomers (2S,3S)-3
and (2S,3R)-3 in a ratio 2.7:1, syn/anti was formed. At this
point, it was not possible to separate these diastereomers
by column chromatography on silica gel. However, the
benzyl group and the PhF protecting group can be
removed in one step by hydrogenation under palladium
catalysis to yield the diastereomeric amino acids 4. At this
point, the diastereomers can be separated on silica gel
using a mixture of ethyl acetate, isopropanol and water
(8:2:1) as eluent. Finally, Fmoc-protection of the amino
group was achieved by treatment of (2S,3S)-b-methyl
aspartic acid tert butyl ester [(2S,3S)-4] and (2S,3R)-b-
methyl aspartic acid tert-butyl ester [(2S,3R)-4] with
Fmoc chloride at 0 ꢀC in dioxane/water using sodium
carbonate as base.
Synthesis
Both b-(2S,3S)- and b-(2S,3R)-methylated N-Fmoc
protected aspartic acid-b tert-butyl ester building blocks
5 were synthesized as shown in Scheme 1 and incorpo-
rated into cyclic RGD peptides. The tert-butyl group as
side-chain protection for the b-carboxyl group of
aspartic acid has proven to be useful in solid-phase
peptide synthesis using the Fmoc strategy. The synthesis
of 5 commences from l-aspartic acid b tert-butyl ester
1, which establishes the desired S configuration of the
a-carbon. Introduction of the bulky 9-phenylfluorene
(PhF) protecting group13 attached to the nitrogen is
achieved by addition of chlorotrimethylsilane, which
protects the a-carboxyl group in situ, and following
reaction with 9-bromo-9-phenylfluorene assisted by
triethylamine and lead nitrate. The TMS-ester is easily
cleaved by aqueous work up. The a-carboxyl group can
then be converted to a-benzyl ester to yield the fully
protected amino acid 2.
Since the coupling constants between the a- and b-sub-
stituted protons, which indicate the stereochemistry,
cannot be differentiated in compounds 4 or in the pro-
tected derivatives 3 or 5 we had to establish the stereo-
chemistry by synthesizing the corresponding alcohols 7
as shown in Scheme 2. To determine the configuration
of the diastereomers the b-methylated diastereomeric
alcohols 7 were synthesized (Scheme 2) starting from a
mixture of diastereomeric aspartic acid methyl ester 6.
The methyl esters 6 were synthesized by reacting
Asp(OMe)OBn with PhF bromide followed by treat-
ment with LiHMDS and methylation of the b-carbon.
The desired compounds were obtained by reduction of
the methyl ester using DIBAL. The diastereomers of 7
were separated and the stereochemistry determined
based on the coupling constants [J(2S,3S), syn=broad s;
J(2S,3R), anti=9.6 Hz) described by Rapoport et al.14
Rapoport and co-workers13a reported the application of
the PhF group and others have recently utilized this
bulky protecting group for its unique properties.13bÀf
Placed on the nitrogen of aspartic acid it shields the
proton on the a-carbon from abstraction by strong
bases. Treatment of 2 with LiHMDS results in the for-
mation of the enolate at b-carbonyl carbon which
allows methylation of the b-position with methyl iodide
Scheme 1. Synthesis of (2S,3R) and (2S,3S) b-methylated aspartic acid building blocks.

S. Schabbert et al. / Bioorg. Med. Chem. 10 (2002) 3331–3337
3333
Scheme 2. Reduction of (2S,3R) and (2S,3S) b-methylated aspartic acid building blocks to alcohols to establish the stereochemistry of the b-carbon.
Reoxidation of the separated alcohols (2S,3S)-7 and
(2S,3R)-7 using PDC in DMF lead to the corresponding
acids 8, which can be converted to the b-tert-butyl ester
by treatment with tert-butyltrichloroacetimidate under
acidic conditions. Cleavage of the a-benzyl ester and
PhF group and subsequent Fmoc-protection of the
amino group yields the desired b-methylated aspartic
acid building blocks 5.
peptides were cleaved from the resin with acetic acid/
trifluoroethanol/dichloromethane 1:1:5 and the peptides
were cyclized in 2.5 mM concentration in DMF using
DPPA. The side chain protecting groups were removed
using trifluoroacetic acid cocktail. Peptide purification
was performed by preparative RP-HPLC on a Vydac
C18column using a gradient of acetonitrile in water
with 0.1% TFA.
Peptide synthesis
Binding data
The peptides were synthesized by stepwise coupling of
Fmoc-amino acid derivatives by using standard cou-
pling procedures on solid-phase Rink amide MBHA
resin for the disulfides or chlorotrityl resin for the head-
to-tail cyclic peptides. The disulfide bonds were formed
on-resin by oxidation of the protected peptides with an
iodine solution in DMF. During this process, the
deprotection of the Acm groups from cysteine and for-
mation of the disulfide bond occurred simultaneously.
Finally, cleavage of the cyclic peptides from the resin as
well as deprotection of all side-chain protecting groups
were accomplished by treatment with a trifluoroacetic
acid cocktail. For the head-to-tail cyclic peptides the
The peptides were assayed for binding to the aIIbb3 and
avb3 receptor using an ELISA assay format as described
in ref 10b. The data as shown in Table 1 demonstrated
clearly that the (2S,3R) b-methyl Asp analogues 9, 11,
and 14 have a considerably lower binding affinity com-
pared to the (2S,3S) b-methyl Asp analogues 10, 12, and
15. The binding affinities of the (2S,3S) b-methyl Asp
analogues 12 and 15 to the avb3 receptor are very simi-
lar to the affinities of the parent compounds 13 and 16.
This is consistent with findings that were reported by
Jackson et al.15 We have carried out NMR studies and
molecular modeling on these peptides which show that
there is no significant difference between the backbone
Table 1. Binding affinities of the (2S,3R) and (2S,3S) b-methylated peptides to the a,b3 and aIibb3 receptors
Peptide sequence
avb3 IC50 (mM)
aIIbb3 IC50(mM)
9
Ac-c[Cys-Arg-Gly-(R)b-MeAsp-Tyr(Me)-Arg-Cys]-NH2
Ac-c[Cys-Arg-Gly-(S)b-MeAsp-Tyr(Me)-Arg-Cys]-NH2
Ac-c[Cys-Arg-Gly-(R)b-MeAsp-Thr-Tic-Cys]-NH2
Ac-c[Cys-Arg-Gly-(S)b-MeAsp-Thr-Tic-Cys]-NH2
Ac-c[Cys-Arg-Gly-Asp-Thr-Tic-Cys]-NH2
2.1
0.03
1.6
4.5
0.15
>10
0.186
n.t.
>10
0228
n.t.
0.167
0.075
0.122
0.75
0.140
10
11
12
13
14
15
16
17
18
19
20
21
0.005
0.005
4.1
0.304
0.181
0.077
0.185
0.002
0.004
0.002
Ac-c[Cys-Arg-Gly-b(R)MeAsp-Asp-Val-Cys]NH2
Ac-c[Cys-Arg-Gly-b(S)MeAsp-Asp-Val-Cys]NH2
Ac-c[Cys-Arg-Gly-Asp-Asp-Val-Cys]NH2
Ac-c[Cys-Arg-Gly-b(S)MeAsp-Asp-(t-BuG)-Cys]-NH2
Ac-c[Cys-Arg-Gly-b(S)MeAsp-b(S)Me Asp-(t-BuG)-Cys]-NH2
c[Arg-Gly-b(S)MeAsp-Asp-(t-BuG)-Mamb]
c[Arg-Gly-b(S)MeAsp-b(S)MeAsp-(t-BuG)-Mamb]
c[Arg-Gly-Asp-Asp-(t-BuG)-Mamb]

3334
S. Schabbert et al. / Bioorg. Med. Chem. 10 (2002) 3331–3337
conformation of the (2S,3R) b-methylated and (2S,3S)
b-methyl Asp containing peptides in a given sequence.
The results of these studies will be reported elsewhere.
overnight at room temperature with shaking. The
receptor solution was removed, and each well was
washed with 200 mL of 0.5% bovine serum albumin in
assay buffer for 10 min. This step was repeated for a
total of three washes. Fifty microliters of 10-fold dilu-
tions (from 0.0002 to 200 mg/mL) of the inhibitory
compounds in assay buffer was added to the wells. Fifty
mL of biotinylated ligand (fibrinogen for aIibb3 and
vitronectin for for avb3) in assay buffer was added to the
wells. The plates were sealed and incubated overnight at
room temperature with shaking.
The differences between the compounds containing the
(2S,3R) b-methylated and (2S,3S) b-methylated Asp
residues can only be attributed to the side-chain orien-
tation of the Asp side chain. Based on these results it
can be assumed that the topochemical array accessible
to the (2S,3S) b-methyl Asp residue is acceptable for
binding to the receptors whereas the (2S,3R) analogues
cannot adopt the Asp side-chain orientation required
for binding.
The ligand/competitor solution was removed, then each
well was washed with 250 mL wash buffer (0.05% Tween
20, 50 mM Tris, 150 mM NaCl2, pH 7.4) for five min.
This step was repeated for a total of three washes. One
hundred microliters of an Avidin Biotin Peroxidase
Complex (Pierce Chemical ABC kit 32050) in wash
buffer was added to each well. The plates were incu-
bated for 30 min at room temperature with shaking.
The ABC solution was removed, then each well was
washed with 250 mL wash buffer for 5 min. This step
was repeated for a total of three washes. One hundred
microliters of a peroxidase substrate (3,3,5,5 tetra-
methylbenzidine, Pierce Chemical TMB substrate kit
34021) was added to each well.
Compounds 17–21 contain two Asp residue in the
sequence and we have incorporated the (2S,3S)
b-methyl Asp residue in one or both of the residues. The
results of this series of compounds demonstrate that the
selectivity of peptides containing the sequence RGDD
can be modified by incorporating a second (2S,3S)
b-methyl Asp residue following the RGD sequence.
When comparing compounds 19, 20, and 21 it is
obvious that compound 19, which contains only one
(2S,3S) b-methyl Asp residue is very similar in potency
and specificity to the parent compound 21. Both com-
pounds are avb3 specific and their binding affinity to the
avb3 receptor is a factor of 50–70 higher than that to the
aIIbb3. The incorporation of a second (S)b-methyl Asp
residue into the this sequence gives rise to a compound
with significantly decreased binding affinity to the aIIbb3.
Compound 20 essentially maintains the binding affinity to
the avb3 receptor. As a result the compound is con-
siderably more avb3 specific than the parent compound
or compound 19. The loss of binding affinity to the aIIbb3
receptor in compound 20 is equivalent to increased avb3
selectivity compared to compound 19 and 21.
The conversion of the substrate was monitored kineti-
cally in a microtiter plate reader (Molecular Devices) at
650 nm. Optical density readings were made of each well
at 12-s intervals for 10 min. The software for the plate
reader was used to calculate the concentration at which
50% of the binding of the ligand to the receptor was
inhibited (IC50). The maximal velocity of the enzymatic
conversion (Vmax) was calculated for each well and
expressed in milli-optical density units per min (mOD/
min). The Vmax values were plotted as a function of
inhibitor concentration, and a four parameter logistic
curve was fitted to the data. The inflection point of this
curve is the IC50.
These findings will be useful in the design of peptido-
mimetics with an appropriate side chain orientation and
increased avb3 selectivity.
Synthesis
Experimental
Abbreviations
N-(9-Phenylfluorenyl)-S-aspartic acid ꢁ-benzyl ꢀ-tert-
butyl ester (2). To a solution of 2.27 g (12 mMol)
l-Asp(OtBu)OH in 25 mL of dry dichloromethane was
added 1.62 mL (1.06 equiv) TMSCl at room tempera-
ture and the mixture was stirred for 2 h. Dry triethyl-
amine (3.56 mL, 2.13 equiv) and, after 15 min, lead
nitrate (3.97 g, 1.0 equiv) and 9-bromo-9-phenylfluorene
(3.85 g, 1.0 equiv) dissolved in 20 mL of dry DCM were
added. Stirring was continued for at least 3 days at rt.
Then methanol (6 mL) was added and after 10 min the
mixture was filtered and evaporated. Aqueous citric acid
and ethyl acetate were added to the residue. After
extraction of the aqueous phase with ethyl acetate the
combined organic layer was washed with brine, dried
and concentrated to give 4.66 g of the PhF-protected
LiHMDS: lithium hexamethyldisilazide; PhF: phenyl-
fluorenyl; Fmoc: fluorenylmethyl oxycarbonyl; DIBAL:
di-isobutylaluminum hydride, PDC: pyridinium dichro-
mate; HOBt: hydroxybenzotriazole; DMF: N,N-dime-
thylformamide; DIEA: diisopropylethyl amine: HATU:
O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium
hexafluorophosphate; HBTU: O - (benzotriazol - 1 - yl) -
N,N,N,N-tetramethyluronium; DIC: diisopropylcarbo-
diimide; TMSCl: trimethylchloro-silane; TFA: trifluoro-
acetic acid; MeCN: acetonitrile, PE: petroleum ether.
Binding assays
1
product N-PhF-Asp(OtBu)OH (90% yield): H NMR
Each well of a microtiter plate (Nunc MaxiSorp) was
coated with 120 mL of purified receptor (0.5 mg/mL in
assay buffer (2 mM CaCl2, 1 mM MgCl2, 50 mM Tris,
150 mM NaCl at pH 7.4) with 4 mM octyl glucoside
(CDCl3, 300 MHz) d 1.42 (s, 9H, tBu), 1.76 (dd, J=17.4,
4.8, 1H, CH2), 2.70 (dd, J=17.4, 3.3, 1H, CH2), 2.80
(dd, J=4.8, 3.3, 1H, CHN), 7.1–7.45 (m, 11H, arom.
H), 7.7–7.8(m, 2H, arom H).

S. Schabbert et al. / Bioorg. Med. Chem. 10 (2002) 3331–3337
3335
The crude N-PhF-Asp(OtBu)OH (2.01 g) was dissolved
in 20 mL DMF and 1.78g (1.05 equiv) Cs CO3 was
ture for 1 h to drive the reaction to completion. After-
wards the solvent was removed. The residue was
dissolved in ethyl acetate, washed with 0.5 M hydro-
chloric acid and brine, dried and concentrated. The
crude product was chromatographed on silica gel using
PE/EA 1:1 as eluent to yield 832 mg (97%) of (2S,3S)-5:
1H NMR (CDCl3, 300 MHz) d 1.25 (d, J=6.9, 3H), 1.44
(s, 9H), 2.97 (m, 1H), 4.23 (t, J=6.9, 1H), 4.40 (d,
J=6.9, 2H), 4.70 (m, 1H), 5.60 (d, J=8.7, 1H), 7.31 (td,
J=7.5, 1.2, 2H), 7.40 (t, J=7.2, 2H), 7.59 (m, 2H), 7.76
(d, J=7.5, 2H). MS (ES) m/e M+Na 448.
2
added at room temperature. After 30 min, benzyl bro-
mide (0.74 mL, 1.2 equiv) was added dropwise and the
mixture was stirred for 3 h. Then the suspension was
filtered through Celite and concentrated under reduced
pressure. Ethyl acetate and satd NaHCO3 solution were
added, the aqueous phase was extracted several times
and the organic phase was washed with brine, dried and
concentrated. The crude product was purified by col-
umn chromatography using PE/EA 10:1 to yield 1.85 g
(68%) of 2 as colorless solid. 1H NMR (CDCl3, 300 MHz)
d 1.44 (s, 9H, tBu), 2.19 (dd, J=15.0, 5.7, 1H, CH2), 2.50
(dd, J=15.0, 5.0, 1H, CH2), 2.98, 3.34 (each m, 2H, CHN,
NH), 4.76, 4.88 (each d, J=12.3, 2H, CH2Ph), 7.1–7.4 (m,
16H, arom H), 7.65 (m, 2H, arom H).
The same procedure was used to synthesize (2S,3R)-5 in
85% yield: 1H NMR (CDCl3, 300 MHz) d 1.24 (d,
J=7.2, 3H), 1.47 (s, 9H), 3.18(m, 1H), 4.24 (t, J=6.9,
1H), 4.40 (m, 2H), 4.57 (m, 1H), 5.86 (d, J=8.7, 1H),
7.31 (td, J=7.5, 1.2, 2H), 7.40 (t, J=7.2, 2H), 7.60 (m,
2H), 7.76 (d, J=7.5, 2H); MS (ES) m/e M+H 426, MN+a
448, (MÀH)À 424.
N-(9-Phenylfluorenyl)-3-methyl-S-aspartic acid ꢁ-benzyl
ꢀ-tert-butyl diester (3). A solution of 1.84 g of 2 in 25
mL of dry THF (5 mL/mmol) was cooled to À78 ꢀC
under nitrogen and 4.6 mL (1.2 equiv) LiHMDS (1 M
solution in THF) was added. After 1 h, 0.29 mL methyl
iodide (1.2 equiv) was added dropwise at À78 ꢀC and
then the mixture was warmed up to room temperature
overnight for completion of the reaction. The solution
was quenched with satd NH4Cl solution. After extrac-
tion with ethyl ether, the combined organic phase was
washed with brine, dried and concentrated under
reduced pressure. The residue was chromatographed on
silica gel (PE/EA 10:1) to give 1.48g of 3 as a solid in
78% yield and a 2.7:1 ratio of syn/anti* diastereomers
N-(9-Phenylfluorenyl)-3(S/R)-methyl-S-homoserine benzyl
ester (7). A mixture of two diastereomers of 6* (1.8:1)
was dissolved in dry DCM (10 mL/mmol) and cooled to
À78 ꢀC under nitrogen. A 1 M solution of DIBAL in
hexane (3 equiv) was dropwise added. After 2 h the
reaction was quenched with methanol and warmed up
to room temperature. The mixture was treated with
ethyl acetate and sat. K/Na-tartrate solution, the aqu-
eous phase was extracted several times with EA and the
combined organic layer was washed with brine, dried
and concentrated. Column chromatography on silica gel
using PE/EA 8:1 as eluent leads to (2S,3S)-7 and
(2S,3R)-7 in 60% yield.
1
according to H NMR (CDCl3, 300 MHz) d 0.87* (d,
J=6.9, 3H, Me*), 1.16 (d, J=7.2, 3H, Me), 1.32, 1.41*
(each s, 9H, tBu), 2.49 (m, each 1H, CHMe), 2.82, 2.98*
(each m, 1H, CHN), 3.10 (m, each 1H, NH), 4.49*,
4.52, 4.68*, 4.70 (each d, J=12.3, OCH2Ph), 7.05–7.4
(m, each 16H, arom H), 7.65 (m, each 2H, arom H).
(2S,3S)-7: 1H NMR (CDCl3, 300 MHz) d 0.87 (d,
J=7.2, 3H, Me), 1.67 (m, 1H, CHMe), 2.64, 3.24 (each
br. s, 1H, NH, OH), 2.83 (br. s, 1H, CHN), 3.37 (dd,
J=11.1, 3.3, 1H, CH2OH), 3.49 (m, 1H, CH2OH), 4.61,
4.78(each d, J=12.3, 1H, OCH2Ph), 7.05–7.46 (m,
16H, arom H), 7.67–7.7 (m, 2H, arom H).
3(2S,3S)- and 3(2S,3R)-Methyl-S-aspartic acid ꢀ-tert-
butyl ester (4). A mixture of 3 (2.27 g) and 10% Pd/C
in 40 mL of methanol was degassed under reduced
pressure and placed under hydrogen (1 atm). After 24 h,
the catalyst was removed by filtration through Celite
and the filtrate was concentrated. Purification and
separation of the diastereomers was achieved on silica
gel using a mixture of ethyl acetate/isopropanol/water
8:2:1 as eluent to yield 410 mg of (2S,3S)-4 and 120 mg
(2S,3R)-4 (88%). TLC ꢀin EA/nBuOH/THF/water 2:2:1:1
Rf 0.27; (2S,3S)-4: [a]2D3 C=À7.0 (c 1, dioxane/water 1:1);
1H NMR (D2O, 400 MHz) d 1.22 (d, J=7.6, 3H, Me),
(2S,3R)-7: 1H NMR (CDCl3, 300 MHz) d 0.48(d,
J=6.9, 3H, Me), 1.91 (m, 1H, CHMe), 2.53 (d, J=9.6,
1H, CHN), 3.37 (dd, J=10.8, 8.7, 1H, CH2OH), 3.56
(dd, J=11.1, 3.0, 1H, CH2OH), 4.33, 4.77 (each d,
J=12.3, 1H, OCH2Ph), 7.07–7.47 (m, 16H, arom H),
7.70 (d, J=7.5, 2H, arom H).
N-(9 -Phenylfluorenyl)-3(S/R)-methyl-S-aspartic acid
benzyl ester (8). To a stirred solution of alcohol
(2S,3S)-7 or (2S,3R)-7 in DMF (10 mL/mmol) were
added 6 equiv PDC at room temperature. After 24 h,
the mixture was diluted with diethylether and washed
with water and brine. The organic layer was dried and
concentrated under reduced pressure. Purification of the
crude product was achieved by column chromatography
on silica gel (PE/EA 3:1).
1.46 (s, 9H, tBu), 3.05 (dq, J=7.6, 4.4, 1H, CHMe), 3.97
ꢀ
(d, J=4.4, 1H, CHNH2). (2S,3R)-4: [a]2D3 C=+13.0 (c 1,
dioxane/water 1:1); 1H NMR (D2O, 400 MHz) d 1.26 (d,
J=7.6, 3H, Me), 1.44 (s, 9H, Bu), 3.13 (dq, J=7.6, 4.4,
1H, CHMe), 3.89 (d, J=4.4, 1H, CHNH2).
t
N-(9-Fluorenylmethoxycarbonyl)-3(S/R)-methyl-L aspartic
acid ꢀ-tert-butyl ester (5). At 0 ꢀC, 535 mg (2.5 equiv)
of sodium carbonate were added to a solution of 410 mg
of (2S,3S)-4 or in 40 mL dioxane/water (2:1). After 15
min 680 mg of Fmoc chloride (1.3 equiv) was added and
stirring was continued for 2 h at 0 ꢀC. Then icebath was
removed and the mixture was stirred at room tempera-
(2S,3S)-8. 55% yield, starting from (2S,3S)-7; 1H NMR
(CDCl3, 300 MHz) d 1.09 (d, J=7.5, 3H, Me), 2.45 (dq,
J=7.5, 4.5, 1H, CHMe), 3.07 (d, J=4.5, 1H, CHN),
4.64, 4.83 (each d, J=12.3, each 1H, OCH2Ph), 7.07–
7.38(m, 16H, arom H), 7.5–7.7 (m, 2H, arom H).

3336
S. Schabbert et al. / Bioorg. Med. Chem. 10 (2002) 3331–3337
(2S,3R)-8. 64% yield, starting from (2S,3R)-7; 1H NMR
(CDCl3, 300 MHz) d 0.82 (d, J=6.9, 3H, Me), 2.53 (dq,
J=10.8, 6.9, 1H, CHMe), 2.83 (d, J=10.8, 1H, CHN),
4.30, 4.77 (each d, J=12.1, each 1H, OCH2Ph), 7.3–7.4
(m, 16H, arom H), 7.70 (m, 2H, arom H).
Ac-c[Cys-Arg-Gly-ꢀ(2S,3R)Me-Asp-Asp-Val-Cys]-NH2.
RP-HPLC: 12.3 min (10–30% MeCN/H2O, 0.1% TFA,
30 min, 10 mL/min); C30H49N11O12S2 (819 g/mol); MS
(ES) m/e M+H 820, MN+a 842, (MÀH)À 818; 1H NMR.
Ac-c[Cys-Arg-Gly-ꢀ(2S,3S)Me-Asp-Thr-Tic-Cys]-NH2.
RP-HPLC: 16.2 min (10–50% MeCN/H2O, 0.1%
TFA, 30 min, 10 mL/min); C35H51N11O11S2 (865 g/
Peptide synthesis
The peptides were synthesized by stepwise coupling of
Fmoc-amino acid derivatives by using standard cou-
pling procedures on solid-phase Rink amide MBHA
resin for the disulfides or chlorotrityl resin for the
homodet cyclic peptides. Usually, HBTU and a 0.5 M
HOBt-solution in DMF were used as activation
reagents for peptide bond formation. The reactions
were carried out in the presence of 2 equiv of DIEA.
For the more hindered coupling to the secondary amino
group in tetrahydroisoquinoline carboxylic acid (Tic)
the more active HATU was utilized instead of HBTU.
The sensitive amino acid cysteine was coupled using a
combination of DIC and 0.5 M HOBt solution without
any base to minimize the risk of racemization. The side-
chain protecting groups were acetamidomethyl (Acm) for
cysteine, the tert-butyl group for aspartic acid and threo-
nine, and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sul-
fonyl (Pbf) for arginine. The cyclic disulfide constraints
in the molecules were formed on-resin by oxidation of
the protected peptides with an iodine solution in DMF.
During this process the deprotection of the Acm groups
from cysteine and formation of the disulfide bond
occurred simultaneously. Finally, cleavage of the cyclic
peptides from the resin as well as deprotection of all side-
chain protecting groups were accomplished by treatment
with a trifluoroacetic acid cocktail, which contains tri-
isopropylsilane, phenol and water as scavengers. For the
head to tail cyclic peptides the peptides were cleaved
from the resin with acetic acid/trifluoroethanol/di-
chloromethane 1:1:5 for 30 min to 1 h. The peptides were
cyclized in 2.5 mM concentration in DMF using 10
equiv of NaHCO3 and DPPA at room temperature. The
side chain protecting groups were removed using tri-
fluoroacetic acid in the presence of triisopropylsilane
(2%), anisole (5%), and water (5%). Peptide purifica-
tion was performed by preparative RP-HPLC on a
Vydac C18column using a gradient of acetonitrile in
water with 0.1% TFA.
mol); MS (ES) m/e M+H 866, (MÀH)À 864; H NMR.
1
Ac-c[Cys-Arg-Gly-ꢀ(2S,3R)Me-Asp-Thr-Tic-Cys]-NH2.
RP-HPLC: 17.1 min (10–50% MeCN/H2O, 0.1%
TFA, 30 min, 10 mL/min); C35H51N11O11S2 (865 g/
mol); MS (ES) m/e M+H 866, (MÀH)À 864.
Acknowledgements
We gratefully acknowledge financial support from the
National Institutes of Health (DK51938-03).
References and Notes
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(Ac-c[Cys-Arg-Gly-ꢀ(2S,3S)Me-Asp-Tyr(Me)-Arg-Cys]-
NH2. RP-HPLC: 18.5 min (10–30% MeCN/H2O, 0.1%
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(MÀTFA)À 1051; H NMR.
Ac-c[Cys-Arg-Gly-ꢀ(2S,3R)Me-Asp-Tyr(Me)-Arg-Cys]-
NH2. RP-HPLC: 19.7 min (10–30% MeCN/H2O, 0.1%
TFA, 30 min, 10 mL/min); C37H58N14O11S2 (938g/
mol); MS (ES) m/e M+H 939, MN+a 961, (MÀH)À 937; 1H
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Ac-c[Cys-Arg-Gly-ꢀ(2S,3S)Me-Asp-Asp-Val-Cys]-NH2.
RP-HPLC: 12.0 min (10–30% MeCN/H2O, 0.1% TFA,
30 min, 10 mL/min); C30H49N11O12S2 (819 g/mol); MS
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